2,4-Thiophenedicarbonitrile, 5-amino-3-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2010 to 18 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

EC Number:

610-868-8

Cas Number:

52603-48-2

Molecular formula:

C7N3H5S

IUPAC Name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 15 - 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum):2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK ad libitum
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: at least five days
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target range of 19 to 25°C
- Humidity (%): target range of 30 to 70%
Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: From: 25 January 2010 To: 18 February 2010

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25%, 10% and 5% w/w in dimethylformamide

No. of animals per dose:

5 per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The vehicle (dimethylformamide) was chosen as it produced the highest concentration that was suitable for dosing (25% w/w).
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 25% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
No signs of systemic toxicity were noted. Beige/light brown coloured residual test material on the ears was noted post dose on Day 3.
Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in dimethyl formamide.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Local Lymph Node Assay in the Mouse.
The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers. The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitisers and non-sensitisers during the in-house validation. The results of the study are believed to be of value in predicting the sensitisation potential of the test material to man.

- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
The test material was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Groups of five mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

Positive control results:

Project number: 0039/1116
Study dates: 11 November 2009 to 17 November 2009

Methods
A group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide alone.

Results
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in Stimulation Index Result
dimethyl formamide
15 5.16 Positive

Conclusion
alpha-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation index per test material concentration: Vehicle control: N/A 5%: 1.12 10%: 1.53 25%: 0.62 Full SI data and interpretations are presented in the table below.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM per test material concentration: Vehicle control: 1425.9 ± 465.52 5%: 1593.12 ± 515.22 10%: 2183.88 ± 932.51 25%: 885.86 ± 272.83 Full DPM data is presented in the table below.

Any other information on results incl. tables

The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given below.

 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (% w/w) in dimethylformamide	Stimulation Index	Result
5	1.12	Negative
10	1.53	Negative
25	0.62	Negative

 

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Beige/light brown coloured residual test material on the ears was noted, post dose on Days 1 to 3, in animals treated with the test material at a concentration of 25% w/w in dimethyl formamide.

 

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

 

Individual Disintegrations per Minute and Stimulation Indices 

Concentration (% w/w) in dimethylformamide	Animal Number	dpm / Animal^a	Mean dpm / Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	1306.14	1425.99 (±465.52)	N/A	Negative
	1-2	1283.11
	1-3	2185.58
	1-4	1434.13
	1-5	921.01
5	2-1	2419.51	1593.12 (±515.22)	1.12	Negative
	2-2	1199.10
	2-3	1361.80
	2-4	1766.38
	2-5	1218.79
10	3-1	3460.45	2183.88 (±932.51)	1.53	Negative
	3-2	2713.87
	3-3	2130.98
	3-4	1423.79
	3-5	1190.29
25	4-1	1094.07	885.86 (±272.83)	0.62	Negative
	4-2	1250.90
	4-3	724.47
	4-4	608.21
	4-5	751.67

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation index of 3.0 or greater indicates a positive result

N/A = Not applicable

 

The results of the statistical analysis of the data indicated there was no significant difference between the control groups and the test groups.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test.

Executive summary:

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in dimethyl formamide at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in dimethylformamide	Stimulation Index	Result
5	1.12	Negative
10	1.53	Negative
25	0.62	Negative

The test material was considered to be a non-sensitiser under the conditions of the test.